The news was announced by the club this morning but it is believed the former Crystal Palace, Barnsley and Millwall Centre Half quit his post last night after a meeting with the board.
Assistant Manager and goalkeeping coach Tony Burns plus former manager Alan McLeary will take charge for The Lions' remaining two games of the season.
"Naturally I am very disappointed that we were not able to avoid relegation," said Tuttle. "But I would like to thank the staff and players for giving it their best shot.
"In particular, I would like to thank the fans for the way they have supported the team during a very frustrating season and I wish the club every success for the future."
Tuttle, who received a vote of no confidence from The Lions Trust last week, is the fourth Millwall manager to leave the post since May, 2005.